The ability to detect alterations in DNA sequences (e.g. mutations and polymorphisms) is central to the diagnosis of genetic diseases and to the identification of clinically significant variants of disease-causing microorganisms. One method for the molecular analysis of genetic variation involves the detection of restriction fragment length polymorphisms (RFLPs) using the Southern blotting technique (Southern, E. M., J. Mol. Biol., 98:503-517, 1975. Since this approach is relatively cumbersome, new methods have been developed, some of which are based on the polymerase chain reaction (PCR). These include: RFLP analysis using PCR (Chehab et al., Nature, 329:293-294, 1987; Rommens et al., Am. J. Hum. Genet., 46:395-396, 1990), the creation of artificial RFLPs using primer-specified restriction-site modification (Haliassos et al., Nucleic Acids Research, 17:3606, 1989), allele-specific amplification (ASA) (Newton CR et al., Nuc. Acids Res., 17:2503-2516, 1989), oligonucleotide ligation assay (OLA) (Landergren U et al., Science 241:1077-1080, 1988), primer extension (Sokolov BP, Nucl. Acids Res., 18:3671, 1989), artificial introduction of restriction sites (AIRS) (Cohen LB et al., Nature 334:119-121, 1988), allele-specific oligonucleotide hybridization (ASO) (Wallace RB et al., Nucl. Acids Res., 9:879-895, 1981) and their variants. Together with robotics, these techniques for direct mutation and analysis have helped in reducing cost and increasing throughput when only a limited number of mutations need to be analyzed for efficient diagnostic analysis.
These methods are, however, limited in their applicability to complex mutational analysis. For example, in cystic fibrosis, a recessive disorder affecting 1 in 2000-2500 live births in the United States, more than 225 presumed disease-causing mutations have been identified. Furthermore, multiple mutations may be present in a single affected individual, and may be spaced within a few base pairs of each other. These phenomena present unique difficulties in designing clinical screening methods that can accommodate large numbers of sample DNAs.
Co-pending U.S. patent application Ser. No. 07/957,205 of Shuber et al. and Shuber et al., Human Molecular Genetics, 2:153-158, 1993, disclose a method that allows the simultaneous hybridization of multiple oligonucleotide probes to a single target DNA sample. By including in the hybridization reaction an agent that eliminates the disparities in melting temperatures of hybrids formed between synthetic oligonucleotides and target DNA, it is possible in a single test to screen a DNA sample for the presence of different mutations. Typically, more than 100 ASOs can be pooled and hybridized to target DNA; in a second step, ASOs from a pool giving a positive result are individually hybridized to the same DNA. Co-pending U.S. patent application Ser. No. 08/281,940 discloses a method for multiple allele-specific disease analysis in which multiple ASOs are first hybridized to a target DNA, followed by elution and sequencing of ASOs that hybridize. This method allows the identification of a mutation without the need for many individual hybridizations involving single ASOs and requires prior knowledge of relevant mutations.
To achieve adequate detection frequencies for rare mutations using the above methods, however, large numbers of mutations must be screened. To identify previously unknown mutations within a gene, other methodologies have been developed, including: single-strand conformational polymorphisms (SSCP) (Orita M et al., Proc. Natl. Acad. Sci. USA 86:2766-2770, 1989), denaturing gradient gel electrophoresis (DGGE) (Meyers RM et al., Nature 313:495-498, 1985), heteroduplex analysis (HET) (Keen j. et al., Trends Genet. 7:5, 1991), chemical cleavage analysis (CCM) (Cotton RGH et al., Proc. Natl. Acad. Sci., 85:4397-4401, 1988), and complete sequencing of the target sample (Maxam AM et al., Methods Enzymol. 65:499-560, 1980, Sanger F. et al., Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977). All of these procedures however, with the exception of direct sequencing, are merely screening methodologies. That is, they merely indicate that a mutation exists, but cannot specify the exact sequence and location of the mutation. Therefore, identification of the mutation ultimately requires complete sequencing of the DNA sample. For this reason, these methods are incompatible with high-throughput and low-cost routine diagnostic methods.
Thus, there is a need in the art for a relatively low cost method that allows the efficient analysis of large numbers of DNA samples for the presence of previously unidentified mutations or sequence alterations.